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A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs
1Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA. yhuang@panda.uchc.edu
Abstract:
Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.
Insights
Mouse polyomavirus mRNA export is hindered by unused 5' splice sites. Splicing is not required for export, and removing splicing machinery may be necessary for efficient mRNA transport.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Mouse polyomavirus serves as a model for mRNA nucleocytoplasmic transport.
- Viral capsid protein mRNAs are generated via alternative splicing.
- Unused splice sites can affect mRNA export efficiency.
Purpose of the Study:
- Investigate the role of unused splice sites in mRNA export.
- Determine if splicing is a prerequisite for mRNA export.
- Identify cis-acting sequences influencing mRNA export.
Main Methods:
- Point mutations and deletion analyses of viral mRNAs.
- Examination of intracellular mRNA distribution.
- RNA stability measurements.
Main Results:
- mVP2 mRNA export efficiency is inversely correlated with 5' splice site strength.
- Unused 3' splice sites have minimal impact on export.
- Splicing does not enhance, and may impede, mRNA export.
Conclusions:
- Suboptimal 5' splice sites significantly influence mVP2 nuclear export.
- Splicing is not essential for polyomavirus mRNA export; removal of splicing machinery might be key.
- Unused splicing signals may affect other viral mRNA export pathways.